1. Field of the Invention
This invention relates to the field of vaccine preparation and, in particular, fermentation of Neisseria bacteria, particularly N. meningitidis, for the production of polysaccharide for use in vaccines.
2. Summary of the Related Art
N. meningitidis causes both endemic and epidemic disease, principally meningitis and meningococcemia. As a result of the control of Haemophilus influenzae type b infections, N. meningitidis has become the leading cause of bacterial meningitis in children and young adults in the United States (US), with an estimated 2,600 cases each year. (Recommendation of the Advisory Committee on Immunization Practices (ACIP). “Control and prevention of meningococcal disease and control and prevention of serogroup C meningococcal disease: evaluation and management of suspected outbreaks.” MMWR 46: No. RR-5, 1997 6 (hereinafter “ACIP”); CDC 1, Laboratory-based surveillance for meningococcal disease in selected areas—United States, 1989-1991, MMWR 42: No SS-2, 1993 (hereinafter “CDC 1”).) The case-fatality rate is 13% for meningitis disease (defined as the isolation of N. meningitidis from cerebrospinal fluid) and 11.5% for persons who have N. meningitidis isolated from blood (ACIP, CDC 1) despite therapy with antimicrobial agents (e.g., penicillin) to which US strains remain clinically sensitive. (ACIP)
Based on multistate surveillance conducted during 1989 to 1991, serogroup B organisms accounted for 46% of all cases and serogroup C for 45%; serogroups W-135 and Y and strains that could not be serotyped accounted for most of the remaining cases. (ACIP, CDC 1) Recent data indicate that the proportion of cases caused by serogroup Y strains is increasing. (ACIP) In 1995, among the 30 states reporting supplemental data on culture-confirmed cases of meningococcal disease, serogroup Y accounted for 21% of cases. (CDC. Serogroup Y Meningococcal Disease—Illinois, Connecticut, and Selected Areas, United States, 1989-1996. MMWR 46:Vol. 45, 1010-1013, 1996 (hereinafter “CDC 2”).) Serogroup A, which rarely causes disease in the US, is the most common cause of epidemics in Africa and Asia. A statewide serogroup B epidemic has been reported in the US. (CDC. Serogroup B meningococcal disease—Oregon 1994. MMWR 44: 121-124,1995 (hereinafter “CDC 3”).) N. meningitidis vaccines comprise group specific polysaccharide antigens. Several discoveries impacted the future of meningococcal polysaccharide vaccines and demonstrated the significance of anti-capsular antibodies in protection. (Frasch, “Meningococcal vaccines; past, present and future,” in Meningococcal Disease, ed. K. Cartwright. John Wiley and Sons Ltd, 1995.) In the late 1930s, serogroup-specific antigens of meningococcal serogroups A and C were identified as polysaccharides. (CDC 3) During the mid 1940s, investigators demonstrated that the protection of mice by anti-serogroup A meningococcal horse serum was directly related to its content of anti-polysaccharide antibodies. (Frasch) Meningococcal polysaccharide vaccines were first demonstrated to be immunogenic in humans by Gotschlich and his co-workers in the 1960s when immunization of US Army recruits with serogroup A and C polysaccharides induced protective antibodies. Id. The investigators recorded a significantly reduced acquisition rate of serogroup C carriage among vaccinated recruits compared with unvaccinated individuals. Id.
Meningitidis polysaccharide manufacture requires fermentation of N. meningitidis. Current good manufacturing practice (cGMP) imposes several criteria to medium development for microbial fermentation for the production of biologics. Ideally, the medium should contain only essential components, be easily prepared in a reproducible manner, and support robust high-cell density culture. A chemically defined medium is inherently more reproducible than a complex medium. Furthermore, a chemically defined medium enables discrete analysis of the effect of each component and strict control of medium formulation through identity and purity testing of raw materials. Finally, the fermentation medium should support the cultivation of the microorganism in question to high-cell density to improve volumetric productivity and to generate a final culture whose composition and physiological condition is suitable for downstream processing.
Catlin [J. Inf. Dis. 128:178-194, 1973], described a complex chemically defined medium named NEDF, containing approximately 54 ingredients, including all twenty naturally occurring amino acids, for growth of Neisseria. In addition, Catlin described a medium called MCDA containing 18 ingredients (in mM: NaCl, 100; KCl, 2.5; NH4Cl, 7.5; Na2HPO4, 7.5; KH2PO4, 1.25; Na3C6H5O7.2H2O, 2.2; MgSO4.7H2O, 2.5; MnSO4.H2O, 0.0075; L-glutamic acid, 8.0; L-arginine.HCL 0.5; glycine, 2.0; L-serine, 0.2; L-cysteine HCl.H2O, 0.06; sodium lactate, 6.25 mg of 60% syrup/mL of medium; glycerin, 0.5% (v/v); washed purified agar, 1% (wt/vol) CaCl2.2H2O, 0.25; Fe2(SO4)3, 0.01) which was reported to support growth of Neisseria meningitidis on agar. The ability of MCDA to support growth in liquid medium (that is absent addition of agar) was not reported. La Scolea et al., [Applied Microbiology 28:70-76, 1974] reported on a defined minimal medium named GGM for the growth of Neisseria gonorrhoeae. The medium contained minimal salts, eight amino acids, two nitrogen bases, vitamins, coenzymes, metabolic intermediates and miscellaneous components. La Scolea et al. reported growth of this strain to an optical density of 400 Klett units. An absorbance of 1 at 600 nm is considered equivalent to 500 Klett units [see Gerhardt et al., Manual of Methods for General Bacteriology, 1981, ASM., p. 197]. Therefore, the maximum reported growth density achieved by LaScolea et al., was less than about one (1) absorbance unit. SU 1750689 A1 described a method for preparing polysaccharide-protein vaccines against Neisseria meningitidis B. A defined medium was described having the following composition, g/L:
Sodium L-glutamate1.30 ± 0.10L-cysteine hydrochloride0.03 ± 0.01Potassium chloride0.09 ± 0.01Sodium chloride6.00 ± 1.00Magnesium sulfate heptahydrate0.06 ± 0.01Ammonium chloride1.25 ± 0.01Disubstituted sodium phosphate2.50 ± 0.20dodecahydrateTrisubstituted sodium citrate0.50 ± 0.10Glucose1.60 ± 0.20
In this medium, it is reported that Neisseria may be cultured to a final optical density of 1.5±0.2 on the FEK-56M scale. This is an unfamiliar scale for optical density determination. However, based on the available carbon sources in the above noted medium, it is predictable that the maximum absorbance achievable would be in the range of about 1.5 absorbance units.
U.S. Pat. No. 5,494,808 reports a large-scale, high-cell density (5 g/L dry cell weight, and an optical density of between about 10-13 at 600 nm) fermentation process for the cultivation of N. meningitidis. This patent disclose the following medium (called “MC.6”) for culturing Neisseria meningitidis for isolation of OMPC (“Outer Membrane Protein Complex”) (all values in mg/L):
NaCl5800K2HPO44000NH4Cl1000K2SO41000Glucose10,000L-Gutamic Acid3900L-Arginine150Glycine250L-Serine500L-Cysteine.HCl100MgCl2.6H2O400CaCl2.2H2O28Fe(III) Citrate40
MENOMUNE® A/C/Y/W-135, Meningococcal Polysaccharide Vaccine, Groups A, C, Y and W-135 Combined, for subcutaneous use, is a freeze-dried preparation of the group-specific polysaccharide antigens from Neisseria meningitidis, Group A, Group C, Group Y and Group W-135. N. meningitidis are cultivated with Mueller Hinton agar1 and Watson Scherp2 media. The purified polysaccharide is extracted from the Neisseria meningitidis cells and separated from the media by procedures which include centrifugation, detergent precipitation, alcohol precipitation, solvent or organic extraction and diafiltration.